Soluble tachykinin antagonists, the preparation and use thereof

ABSTRACT

The compounds of formula (I) cyclo[X 1   1  --Z 1   2  --X 2   3  --X 3   4  --Z 2   5  --X 4   6  ]cyclo(2β-5β) wherein: X 1  =(D or L)Cys(Y) or (D or L)SeCys(Y), Z 1  =Asp and Z 2  =Dap or Z 1  =Dap and Z 2  =Asp and X 2 , X 3  and X 4  =natural or synthetic hydrophobic amino acids, having Z 1 , Z 2 , X 2 , X 3  and X 4  the same D or L configuration; Y=is a glycosidic group selected from the aldo and keto hexoses in the furanose or pyranose form bound to the cysteine with an α or β thioacetalic bond or a cyclithol or a polyvinyl alcohol or PEG, constituted of 5 to 10 monomeric unities, bound to the cysteine with a thioether bond, are endowed of high solubility and of potent tachykinin-antagonistic activity.

The present invention refers to soluble tachykinin antagonists, to theirpreparation and to pharmaceutical compositions containing them.

Many tachykinin antagonists are known in literature and among these themonocycle (A. T. McKnight et al., Br. J. Pharmacol. 1991, 41,376) andbicycle (V. Pavone et al., J. Chem. Soc. Perkin Trans 2, 1995, 987)compounds are particularly interesting. All the compounds developed upto now are characterized by a high hydrophobicity and therefore are allwater insoluble. In fact patent WO 93/21227 discloses insoluble bicyclepeptide tachykinin antagonists, wherein all the claimed structures arecharacterized by amino acids with hydrophobic side chains or if thechains are polar, they are functionalized so as to confer hydrophobicityto the functional group. The low water solubility (no more than 15μg/ml) which inevitably characterizes these extremely hydrophobiccompounds, has effectively hampered any possible pharmacologicalapplications of such molecules up to now, notwithstanding their in vitrointeresting biological activity. It is likewise known from literature(L. Quartara et al., J. Med. Chem. 1994, 37, 3630) that an increase inthe hydrophilicity of this class of molecules causes a progressivereduction in their biological activity. From this reference point, thepharmaceutical industry's interest comes out in the search for newclasses of tachykinin-antagonists of the water soluble type, but with ahighly specific activity.

The present invention discloses tachykinin-antagonists which at the sametime present a high water solubility and a high biologic activity. Thecompounds of the invention have the following general formula (I):cyclo[X₁ ¹ --Z₁ ² --X₂ ³ --X₃ ⁴ --Z₂ ⁵ --X₄ ⁶ ]cyclo(2β-5β) wherein: X₁=(D or L)Cys(Y) or (D or L)SeCys(Y), Z₁ =Asp and Z₂ =Dap or Z₁ =Dap andZ₂ =Asp and X₂, X₃ and X₄ =natural or synthetic hydrophobic amino acids,having Z₁, Z₂, X₂, X₃ and X₄ the same D or L configuration; Y=is aglycosidic group selected from the aldo and keto hexoses in the furanoseor pyranose form bound to the cysteine or to the selenocysteine with anα or β thio- or seleno-acetal bond or a cyclithol or a polyvinyl alcoholor PEG, constituted of 5 to 10 monomeric unities, bound to the cysteineor to the seleno cysteine respectively with a thio- or seleno-etherbond.

The compounds with X₂ =Trp or 1-Nal; X₃ =Phe and X₄ =Leu or Cha areparticularly preferred.

The compounds of formula (I) have many chiral centres, and therefore theinvention comprises all the possible enantiomers.

The compounds of formula (I) show unique structural characteristics,which differentiate them from the known products. In fact, the claimedcompounds, contrary to those reported up to now (A. T. McKnight et al.,Br. J. Pharmacol. 1991, 41, 376; C. A. Maggi et al., J. Pharmacol. andExpt. Therapeutics, 1994, 271, 1489), contain highly hydrophilicfunctional groups, which give them an at least 100 times higher watersolubility. The high water solubility and the strong pharmacologicalactivity, higher than that of the insoluble compounds previouslydeveloped, make this new class of molecules ideal for the development ofa wide set of pharmaceutical formulations. As far as these applicativeaspects are concerned, the compounds of the invention substantiallydiffer from the insoluble derivatives previously studied in that thelatter do not permit the development of pharmaceutical compositionswhich can be used therapeutically.

In particular, the compounds of formula I characterized by a residue ofcysteine or of selenocysteine, whose respective --SH or --SeH group isinvolved in a thio- or seleno-glycosidic or thio- or seleno-ether bondwith a galactose, glucose, inositol or PEG residue or low molecularweight polyalcohols, even though with high water solubility, they havean activity even a little higher than the activity of the bestantagonists of the insoluble type, such as the MEN 10627 compound (C. A.Maggi et al., J. Pharmacol. and Expt. Therapeutics, 1994, 271, 1489),which has a solubility of only 15 μg/ml. This is even more surprising ifit is considered that up to now the data available in literature showedthat when the water solubility increases inevitably the pharmacologicalactivity of such compounds decreases (L. Quartara et al., J. Med. Chem.1994, 37, 3630).

The compounds of formula (I) behave in a surprising opposite manner withrespect to the analogues wherein cysteine functionalized withβ-D-galactose has been substituted with the following residues:

Asn(β-D-gal): Gln(β-D-gal); Ser(β-D-gal); hSer(β-D-gal); hCys(β-D-gal).

In particular, the analogues with Asn(β-D-gal) and Ser(β-D-gal) weresynthesized for comparison and the solubility and biologic activity arereported in the example 2.

The compounds of formula (I), which are the object of the presentinvention, can be synthesized according to the different techniquesknown from literature, e.g. Schroeder et al. "The Peptides" vol. 1,Academic. Press., 1965; Bodanszky et al. "Peptide Synthesis"Interscience Publischer, 1966; Barany & Merrifield, "The peptides;Analysis, Synthesis, Biology", 2, Chapter 1, Academic. Press., 1980.These techniques include the peptide-solid phase synthesis, the peptidesynthesis in solution, the synthetic methods of the organic chemistry,or any combination thereof. The chosen synthetic scheme will obviouslydepend on the composition of the particular molecule. Since all of theclaimed molecules are peptides, synthetic methods based on appropriatecombinations of solid phase techniques and the classic methods insolution are preferably used, which involve low costs of productionparticularly in an industrial scale. In details such methods consist in:

i) the synthesis in solution of fragments of the peptide chain throughthe sequential coupling of the N-protected amino acids, appropriatelyactivated, with an amino acid or with a C-protected peptide chain, withisolation of the intermediates, following selective deprotection of theN and C terminals of said fragments and their coupling till thepreparation of the desired peptide. Finally the groups involved in thecyclizations, for instance the N and C terminals or the side chains, areselectively deprotected and afterwards are condensed. Finally, ifnecessary, the side chains are deprotected.

ii) The solid phase synthesis of the peptide chain from the C-terminalto the N-terminal on an insoluble polymeric support, the cyclization insolid phase between the side chains previously deprotected. The peptideis removed from the resin through the hydrolysis with anhydroushydrofluoric acid or with trifluoroacetic acid in the presence ofsuitable scavengers and then the cyclization of the monocyclic peptideis carried out in diluted solution. Then the side chains aredeprotected.

Fmoc-Cys(β-D-Gal(Ac)4)-OH, Fmoc-Ser(β-D-Gal(Ac)4)-OH,Fmoc-Asn(β-D-Gal)-OH, can be obtained according to the methods known inliterature (M. Gerz, et al., Angew. Chem. Int. Ed. Engl., 1993, 32, 269;Kessler, H. et al. J. Am. Chem. Soc. 1992, 114, 4805; Kessler H. et al.,J. Am. Chem. Soc. 1991, 113, 7550).

The seleno-derivatives are obtained with similar procedures startingfrom the selenocysteine, using the known techniques for the protectionof the amino function.

The above indicated compounds of formula (I) resulted in more potentantagonists than other analogous antagonists and thus they can beadministered in amounts lower than those required for the knownproducts.

Thus these compounds are suitable for the therapeutic administration tothe superior animals and to humans. In fact their water solubilityallows to prepare simple and unexpensive pharmaceutical preparations,suitable for the parenteral, oral, inhalatory and sublingualadministrations, while their high biologic activity allows to obtaineffective pharmacological effects even with low amounts. Examples ofsuitable formulations comprise capsules, tablets, syrups, solutions orsterile injectable lyophils, aerosols.

The compounds of the invention can be administered in amounts comprisedbetween 0.01 mg/Kg and 10 mg/Kg for the treatment of arthritis, asthma,inflammation, tumoral growth, gastro intestinal hypermotility,Huntington disease, neuritis, neuralgia, migraine, hypertension,incontinence of urine, urticaria, symptoms of the carcinoid syndrome,influenza and cold.

The following examples which are not limitative further illustrate thecompounds of the invention.

List of the Abbreviations

The nomenclature and abbreviations of the amino acids are consistentwith the recommendations of the IUPAC-IUB Joint Commission onBiochemical nomenclature (Eur. J. Biochem. 1984, 138:9); the amino acidsare meant in the L configuration if not otherwise specified. The otherabbreviations are: NKA=neurokinin A; SP=substance P;Dap=2,3-diaminopropionic acid; SeCys=selenocysteine; PEG=polyethyleneglycol; 1-Nal=1-naphthyl-alanine; Cha=cyclohexylalanine; gal=galactose;hSer=homoserine; hCys=homocysteine; Boc=tert-butyloxycarbonyl;PAM=phenylacetamidomethyl; Fmoc=9-fluorenylmethyloxycarbonyl;PyBop=benzotriazol-1-yl-oxypyrrolidinephosphonium hexafluorophosphate;DIEA=diisopropylethylamine; Fm=fluorenylmethyl;DCC=dicyclohexylcarbodiimide; DMF=N-N' dimethylformamide;DCM=dichloromethane; i-PrOH=isopropanol; Ac=acetyl; DMS=dimethylsulfur;MeOH=methanol; Rt=retention time; FAB-MS=fast atom bombardment massspectrometry; HPLC=high pressure liquid chromatography;HOBt=1-hydroxybenzotriazole; TFA=trifluoroacetic acid.

EXAMPLE 1

A) Preparation of the compound of general formula I wherein X₁=Cys(β-D-Gal); Z₁ =Asp; X₂ =Trp; X₃ =Phe; Z₂ =Dap; X₄ =Leu {cyclo[Cys¹(β-D-Gal)-Asp² -Trp³ -Phe⁴ -Dap⁵ -Leu⁶ ]cyclo(2β-5β)}(SEQ ID NO:1).

The peptide was assembled in solid phase, through Boc chemistry, on aPAM resin functionalized with Boc-Leu, using a ABI 430A AppliedBiosystem synthesizer. The standard deprotection and coupling cycleswere used. The following amino acids were coupled in the order with0.651 g of Boc-Leu-PAM resin (scale 0.5 mmol): Boc-Dap(Fmoc)-OH (1.5mmol; PyBop 1.5 mmol, DIEA 3 mmol); Boc-Phe-OH, Boc-Trp(CHO)-OH andBoc-Asp(OFm)-OH (2 mmol and DCC/HOBt as the coupling agent). Beforecarrying out the cyclization between the β-carbonyl of Asp and theβ-amino group of Dap, the side chains of the two amino acids weredeprotected using two deprotection cycles with a solution 20% piperidinein DMF of 3 and 7 min respectively. Repeated washings were then carriedout with the following solvents: DCM, DMF, DCM, and i-PrOH. Thecyclization was carried out adding PyBop (1.5 mmol) and DIEA (3 mmol) tothe suspended resin in 15 ml of DMF. The solution was reacted overnight.Afterwards washing cycles were carried out using DMF and DCM. After theAsp amino group deprotection, Fmoc-Cys(β-D-Gal(Ac)4)-OH (1.5 mmol) wascoupled to the monocyclic peptide using PyBop as coupling agent (0.78 g,1.5 mmol) and DIEA (3 mmol) as the base. The reaction was carried outovernight. The Cys-Fmoc group deprotection was carried out as describedfor the side chains of Dap and Asp. The removal of the monocyclicpeptide from the resin was carried out according to the low/high HFprocedure described by Tam & Merrifield (HF/DMS/p-thiocresol/p-cresol2.5/6.5/0.5/0.5; 4 h at 0° C., followed by HF/p-cresol 9/1; 30 min at 0°C. 362 mg of crude product were recovered (67% yield), which werepurified by preparative HPLC yielding 100 mg of pure product (20%yield), in accordance with the analytical HPLC (Rt=17.7 min;purity>99%). 48 mg of PyBop (1 eq.) and 46 μl of DIEA (3 eq.) pH 8-8.5were added to 100 mg of the peptide (3) (0.0926 mmol) which weredissolved in 93 ml of DMF (1 mM). The solution was reacted overnight,the completeness of the reaction was determined by analytical HPLC.After eliminating DMF, the sample was dissolved in CHCl₃ and extractedtwo times with a saturated NaHCO₃ solution and two times with water. Theorganic phase was dried over Na₂ SO₄ and the solvent was removed undervacuum. The crude material was redissolved in CH₃ CN/water andlyophilized. 85.1 mg (87%), HPLC (Rt=19.9 min; purity>88%) wereobtained. CH₃ ONa (1 eq; 346 μl of a solution containing 5 mg/ml of Nain MeOH) was added to a solution containing 80 mg of (4) (0.0753 mmol)in 64 ml of anhydrous MeOH (1.25 mg peptide/ml MeOH). The reaction wascompleted in 90 min and the progress was controlled through analyticHPLC. The solution was dried and the product purified by preparativeHPLC. The crude product showed a single peak (Rt=14.9 min; area>80%).After purification on preparative HPLC, 29.6 mg with Rt=14.9 min(purity>94%) were obtained. The FAB-MS shows the presence of themolecular ion MH⁺ =895 amu, corresponding to the desired product.

Comparative Example 1

Preparation of the compound of general formula (I) wherein X₁=Ser(β-D-Gal); Z₁ =Asp; X₂ =Trp; X₃ =Phe; Z₂ =Dap; X₄ =Leu {cyclo[Ser¹(β-D-Gal)-Asp² -Trp³ -Phe⁴ -Dap⁵ -Leu⁶ ]cyclo(2β-5β) (SEQ ID NO:2).

The peptide was assembled in solid phase, through the Boc chemistryaccording to a procedure identical to the procedure for the cyclo[Cys¹(β-D-Gal)-Asp² -Trp³ -Phe⁴ -Dap⁵ -Leu⁶)cyclo(2β-5β) compound (SEQ IDNO:1), using Fmoc-Ser¹ (β-D-Gal-(Ac)₄)-OH instead of Fmoc-Cys¹(β-D-Gal(Ac)₄)-OH. The bicyclic crude product showed a single peak(Rt=14.0 min; purity>85%). After purification on preparative HPLC 31 mgwere obtained with Rt=14.0 min (purity>94%). The FAB-MS analysis showsthe presence of the molecular ion MH⁺ =879 amu, corresponding to thedesired compound.

Comparative Example 2

Preparation of the compound of general formula I wherein X₁=Asn(β-D-Gal); Z₁ =Asp; X₂ =Trp; X₃ =Phe; Z₂ =Dap; X4=Leu {cyclo[Asn¹(β-D-Gal)-Asp² -Trp³ -Phe⁴ -Dap⁵ -Leu⁶ ]cyclo(2β-5β)) (SEQ ID NO:3).

The peptide was assembled in solid phase, through the Fmoc chemistry, ona Macrosorb SPR resin functionalized with the p-hydroxymethylbenzoicacid, a base labile linker, using a Milligen 9010 synthesizer. Thestandard deprotection and coupling cycles were used.

The esterification of the first residue was carried out with 0.6 mmol(triple excess) of (Fmoc-Leu)₂ O in DMF. The coupling ofFmoc-Dap(Boc)-OH, Fmoc-Phe-OH, Fmoc-Trp-OH and Fmoc-Asp(OtBu)-OH wascarried out with 0.8 mmol of the amino acid and HOBT/DCC as theactivating agent. Before carrying out the cyclization between theβ-carbonyl of Asp and the β-amino group of Dap, the deprotection of thetwo amino acidic side chains was carried out with a solution TFA/H₂ O(90:10). The cyclization was performed by adding HOBt/DCC (1.6 mmol) tothe resin suspended in DMF. The reaction was carried out overnight.After the Asp-amino group deprotection, Fmoc-Asn(β-D-Gal)-OH (0.8 mmol)was coupled to the peptide using PyBop (0.8 mmol) as the coupling agentand DIEA (1.2 mmol) as the base. The reaction was carried out overnight.The removal of the monocyclic peptide from the resin was carried out byadding 10 ml of a 1 M NaOH solution to 1 g of peptidyl-resin at 4° C.The reaction was effected for 15 min at room temperature. The solutionwas filtered, and the filtrate was collected into a beaker containing 10ml of an aqueous solution of 10% acetic acid. The resin was washed withwater and the pH of the filtrate adjusted at 7 with acetic acid. 48 mgof PyBop (1 eq.) and 46 μl of DIEA (3 eq.) were added to 0.1 mmol of thepeptide (3) dissolved in 93 ml of DMF (1 mM), keeping the pH between 8and 8.5. The solution was reacted overnight, the progress of thereaction was determined through analytic HPLC. The solution was driedand the product purified with preparative HPLC. The crude product showeda single peak (Rt=13.5 min; purity>80%). After purification onpreparative HPLC, 25 mg with Rt=13.5 min (purity>94%) were obtained. TheFAB-MS analysis shows the presence of the molecular ion MH⁺ =992 amu,corresponding to the desired compound.

EXAMPLE 2 In vitro Biologic Activity

The capability of the products of the present invention of acting asantagonists of the neurokinin A receptor was evaluated using apreparation in which the biologic response produced by tachykinins andby the correlated peptides is determined exclusively by the neurokinin Areceptor (NK-2 receptor). The deferent conduit, which is contracted bythe tachykinins in a dose-dependent manner, was used as the experimentalmodel. The determination of the peptide activity was carried out using aconcentration of β-Ala⁸ -NKA[4-10] (3 nM), as the agonist, whichproduces a response equal to 45% of the highest response. The activityof the peptides described in the example 1 was evaluated for theinhibition of the response to the agonist, as a function of theconcentration, and compared to the insoluble MEN-10627 compound (V.Pavone et al., J. Peptide Science 1, 236). The table reports the valuesof pA2 and the solubility of the compounds.

    ______________________________________                                                                  Solubility                                          Compound           pA.sub.2                                                                             μg/ml                                            ______________________________________                                        Example 1          8.4    1.800                                               Comparative example 1                                                                            6.2    2.020                                               Comparative example 2                                                                            5.8    1.980                                               MEN 10627          8.1    15                                                  ______________________________________                                    

What is claimed is:
 1. Compounds of general formula (I) cyclo[X₁ ¹ --Z₁² --X₂ ³ --X₃ ⁴ --Z₂ ⁵ --X₄ ⁶ ]cyclo(2β-5β) wherein: X₁ =(D or L)Cys(Y)or (D or L)SeCys(Y), Z₁ =Asp and Z₂ =Dap or Z₁ =Dap and Z₂ =Asp and X₂,X₃ and X₄ =natural or synthetic hydrophobic amino acids, having Z₁, Z₂,X₂, X₃ and X₄ the same D or L configuration; Y=is a glycosidic groupselected from the aldo and keto hexoses in the furanose or pyranoseform, bound to the cysteine through an α or β thioacetalic bond or acyclithol or a polyvinyl alcohol or PEG, constituted by 5 to 10monomeric units, bound to the cysteine with a thioether bond. 2.Compounds according to claim 1 wherein: X₁ =(D or L)Cys(Y) or (D orL)SeCys(Y), Z₁ =Asp and Z₂ =Dap or Z₁ =Dap and Z₂ =Asp and X₂ =Trp or1-Nal, X₃ =Phe and X₄ =Leu or Cha, having Z₁, Z₂, X₂, X₃, and X₄ thesame D or L configuration and Y=being defined as in claim
 1. 3.Pharmaceutical compositions containing a compound of claim 1, mixed witha suitable vehicle.